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Cellecta provides construction of custom lentiviral constructs expressing shRNA targeting any transcript.
Cellecta offers cloning of specific hairpin sequences into one of our standard or customized shRNA lentiviral expression vectors; or we will design and clone 3 to 5 shRNA constructs targeting any human or mouse gene.
Following cloning, constructs are sequenced and we provide you with lentiviral plasmid DNA and, optionally, pre-packaged, ready-to-transduce lentiviral particles.
Our lentiviral service is typically used by researchers who have found hits using a Cellecta pooled shRNA library, or who are interested in knocking down a specific gene. You can provide us with the hairpin sequences you want to clone, the reference for particular shRNAs in one of our libraries, or just ask us to design a few constructs to your target of interest. We offer a range of vectors with different fluorescent and selection markers with which to make constructs. We will synthesize the DNA to encode the desired hairpin, clone it, and sequence it to verify correct construction. Optionally, we offer packaging services to produce lentiviral particles that may be used to directly transduce your cells of interest.
Overall, about 70% of our constructs knock down the target by more than 70% based on qRT-PCR, so it is likely that at least 2 of 3 or 3 of 5 constructs will be effective. However, we do not guarantee this level for any specific target, and the percentage of highly effective shRNAs will vary from target to target. Also, you may see a phenotype with just a small percent knockdown or require a much higher knockdown before a phenotype manifests itself, depending on the nature of the gene target. If you choose to have us design the sgRNA sequences, you will receive several of our best-designed constructs to test and characterize so you can determine which of these best match your experimental needs.
RNAi provides an approach to directly knock down the level of a gene transcript. This technology, in contrast to CRISPR sgRNA which knocks out a gene target at the genomic level, does not alter the genomic DNA. Also, shRNA knockdown typically occurs more rapidly than CRISPR knockout and is reversible--when the shRNA is removed, expression levels of the target gene return to normal. Finally, RNAi knockdown makes use of an endogenous pathway in mammalian systems, so only expression of the shRNA is required, unlike with CRISPR where co-expression of the Cas9 nuclease is necessary for the system to work.
These features that differentiate RNAi-based shRNA knockdown from CRISPR make it suitable for a number of application where CRISPR might be inconvenient (e.g., in vivo use, use in primary cells where it is difficult to express Cas9, etc.) or where complete knockout of a target is problematic, such as with highly essential genes or when the interest is to mimic a drug compound effect, which are typically inhibitors.
The reversibility of shRNA can also be useful for some studies. Inducible shRNA expression is often useful to demonstrate that a phenotype (e.g., cell viability) is linked to expression of the shRNA. The phenotype appears when an shRNA is expressed and disappears when the same shRNA is repressed. To facilitate these types of analyses, we offer constructs with inducible versions of both the H1 and U6 RNA polymerase III promoters using the tetracycline repressor element. The addition of tetracycline (actually, a tetracycline-analog doxycycline) induces expression of the shRNA.
